ERp44/CG9911 promotes fat storage in Drosophila adipocytes by regulating ER Ca2+ homeostasis.

Fat storage is one of the important strategies employed in regulating energy homeostasis. Impaired lipid storage causes metabolic disorders in both mammals and Drosophila. In this study, we report CG9911, the Drosophila homolog of ERp44 (endoplasmic reticulum protein 44) plays a role in regulating adipose tissue fat storage. Using the CRISPR/Cas9 system, we generated a CG9911 mutant line deleting 5 bp of the coding sequence. The mutant flies exhibit phenotypes of lower bodyweight, fewer lipid droplets, reduced TAG level and increased expression of lipolysis related genes. The increased lipolysis phenotype is enhanced in the presence of ER stresses and suppressed by a reduction of the ER Ca2+. Moreover, loss of CG9911 per se results in a decrease of ER Ca2+ in the fat body. Together, our results reveal a novel function of CG9911 in promoting fat storage via regulating ER Ca2+ signal in Drosophila.

Loss of Dysbindin Implicates Synaptic Vesicle Replenishment Dysregulation as a Potential Pathogenic Mechanism in Schizophrenia.

The schizophrenia-susceptibility gene, dystrobrevin-binding protein 1 (DTNBP1), encodes the dysbindin protein and mediates neurotransmission and neurodevelopment in normal subjects. Functional studies show that DTNBP1 loss may cause deficient presynaptic vesicle transmission, which is related to multiple psychiatric disorders. However, the functional mechanism of dysbindin-mediated synaptic vesicle transmission has not been investigated systematically. In this study, we performed electrophysiological recordings in calyx of Held synapses. We found that excitatory postsynaptic current (EPSC) and miniature EPSC (mEPSC) amplitudes were unchanged in dysbindin-deficient synapses, but readily releasable pool (RRP) size and calcium dependent vesicle replenishment were affected during high-frequency stimulation. Moreover, dysbindin loss accompanied slightly decreases in Munc18-1 and snapin expression levels, which are associated with vesicle priming and synaptic homeostasis under high-frequency stimulation. Together, we inferred that dysbindin directly interacts with Munc18-1 and snapin to mediate calcium dependent RRP replenishment. Dysbindin loss may lead to RRP replenishment dysregulation during high-frequency stimulation, potentially causing cognitive impairment in schizophrenia.

Rosuvastatin stabilizes atherosclerotic plaques by reducing CD40L overexpression-induced downregulation of P4Hα1 in ApoE-/- mice.

Background Cluster of differentiation 40 ligand (CD40L) and rosuvastatin (RSV) affect atherosclerotic plaque stability, but little is known about their roles in extracellular matrix (ECM) production. We investigated the effects of CD40L and RSV on pre-existing advanced plaques. Methods and results Pre-existing advanced plaques were induced in apolipoprotein E-knockout (ApoE-/-) mice by the surgical placement of carotid constrictive silastic collars. Two weeks after surgery, mice were divided into the following treatment groups: control, empty adenovirus, CD40L adenovirus, CD40L adenovirus + RSV, and RSV. Mice received adenovirus via two tail-vein injections (2 × 109 pfu each) and/or RSV via intragastric administration (5 mg/kg; daily for 4 weeks). Mice in the CD40L adenovirus group exhibited increased plaque disruption rates, increased relative plaque macrophage and lipid content, reduced plaque collagen content, and increased local inflammation compared to the other treatment groups, but no significant differences in plaque area were observed among the groups. Notably, in the atherosclerotic plaques of the CD40L adenovirus group, both the mRNA and protein expression of prolyl-4-hydroxylase alpha 1 (P4Hα1) was significantly decreased, leading to a consequent decrease in the protein expression of collagen types I and III. Treatment with RSV decreased the serum levels of CD40L in a lipid-independent fashion and attenuated the effects of CD40L overexpression, particularly with respect to P4Hα1 downregulation. Conclusions CD40L destabilized advanced plaques in the carotid arteries of ApoE-/- mice, in part by decreasing P4Hα1 expression, and consequently collagen expression. These destabilizing effects were attenuated by RSV.

Research on the pharmacodynamics and mechanism of Fraxini Cortex on hyperuricemia based on the regulation of URAT1 and GLUT9.

Fraxini Cortex (also known as Qinpi, QP) has been used for the treatment of hyperuricemia with a significant difference on efficacy of QP from different regions. However, it`s still unknown whether proportion of components is the key and why same kind of herbs have different therapeutic effects. In this study, different sources of QP were collected from Shaanxi Qinpi extracts (SQPE), Henan Qinpi extracts (HQPE), Hebei Qinpi extracts (GQPE) provinces in China. Rat model of hyperuricemia with hypoxanthine combined with potassium oxonate were established to determine the levels of blood urea nitrogen (BUN), serum uric acid (SUA), urine uric acid (UUA) and creatinine (Cr). Hematoxylin-eosin staining (H&E) and Periodic Acid-Schiff staining (PAS) were performed for renal pathology while Western blot analysis and real-time PCR analysis for proteins and mRNA expression levels. High-performance liquid chromatograph (HPLC) was used for components and composition analysis. Our results demonstrated that QPE from different regions could alleviate hyperuricemia via increasing significantly the SCr and BUN levels whereas decreasing markedly UCr, SUA and UUA levels. Additionally, QPE could also improve the pathological changes of the kidneys. The protein and mRNA levels of urate reabsorption transporter 1 (URAT1) and glucose transporter 9 (GLUT9) were down-regulated by QPE treatment. SQPE hold a better activity on improving hyperuricemia and regulating URAT1 and GLUT9. HPLC analysis showed that the proportion of four components aesculin, aesculetin, fraxin, fraxetin were 9.002: 0.350: 8.980: 0.154 (SQPE); 0.526: 0.164: 7.938: 0.102 (HQPE); 12.022: 1.65: 0.878: 1.064 (GQPE). These data indicate that this proportion of effective components may be an important factor for efficacy of QP and had implications for the treatment of hyperuricemia.

Regulation of Eclipta prostrata L. components on cigarette smoking-induced autophagy of bronchial epithelial cells via keap1-Nrf2 pathway.

Cigarette smoking extract (CSE)-induced autophagic injury has been regarded as an important contributor to the pathogenesis of lung cancer. We previously found that Eclipta prostrata L. component (CCE) reduced CSE-induced bronchial epithelial cells damage. However, the mechanism remains unknown. Human normal bronchial epithelial cells (NHBE) were exposed to CSE to establish stress model. Nrf2-siRNA and Keap1-siRNA transfection were performed. mRFP-GFP-LC3 dual fluorescence and transmission electron microscopy were used to observe the autophagic characteristics. CCE prevented CSE-induced Nrf2 transfer into cytoplasm and up-regulated Keap1 level of NHBE cells. Furthermore, CCE significantly increased p-p16, p-p21 and p-p53 phosphorylation levels in Nrf2-siRNA- or Keap1-siRNA-transfected cells. As demonstrated by transmission electron microscopy and mRFP-GFP-LC3 dual fluorescence assays, CCE mitigated autophagic injury, and also down-regulated autophagy-related Beclin-1, LC3II/LC3I ratio, Atg5 and ATF4 levels. Our findings showed the attenuation of CCE on CSE-induced NHBE cells injury was associated with Nrf-2-mediated oxidative signaling pathway.

Lactone Component From Ligusticum chuanxiong Alleviates Myocardial Ischemia Injury Through Inhibiting Autophagy.

The dysregulation of autophagy is associated with a series of cardiovascular diseases, such as myocardial ischemia injury. Lactone component from Ligusticum chuanxiong (LLC) is the major constituent of the traditional Chinese herb L. chuanxiong Hort., which has been reported to hold potential cardioprotective effects. In this study, to determine whether LLC protects the heart through regulation of autophagy, we explored the effects of LLC on cardioprotection and autophagy in myocardial ischemia injured rats and H9c2 cardiomyocytes. Our results showed that LLC significantly reduced infarct size and serum levels of lactate dehydrogenase, creatine kinase, and cardiac troponin and ameliorated histological features in a dose-dependent manner. Similar protections were observed in cardiomyocytes subjected to oxygen-glucose deprivation (OGD). Meanwhile, LLC inhibited autophagy induced by myocardial ischemia injury, characterized by increased autophagic vacuoles, LC3-II/LC3-I ratio and the expression of Beclin 1, whereas decreased the expression of p62. Additionally, LLC combined with a lysosomal inhibitor chloroquine (CQ) reduced LC3-II/LC3-I ratio in cardiomyocytes compared with CQ alone. Furthermore, LLC-afforded cardioprotection was abolished by a specific PI3K inhibitor LY294002. Collectively, these findings demonstrated that cardioprotective effects of LLC were related to restoration of autophagic flux through the activation of PI3K/Akt/mTOR signaling pathway.

Autophagy Flux Contributes to Regulation of Components of Eclipta prostrata L. on Cigarette Smoking-Induced Injury of Bronchial Epithelial Cells.

Excessive autophagy plays a crucial role in cigarette smoking extract (CSE)-induced inflammation response and oxidative damage of respiratory epithelial cells. The components from Eclipta prostrata L. (CCE) have been shown to be beneficial for CSE-induced epithelial cells injury. However, whether its protection on CSE-stress injury is related to its regulation on autophagy remains still unclear. In this study, CCE, containing mainly wedelolactone of 45.88% and demethylwedelolactone of 23.74%, could improve significantly 10%CSE-induced cell viability of normal human bronchial epithelial (NHBE) cells using CCK-8 kit. We revealed that CCE could remarkably increase autophagic factors Beclin-1, Atg5, ATF4 proteins expression levels and the transformation of LC3-I to LC3-II. Additionally, CCE up-regulated significantly p-p16 and p-p21 phosphorylation levels whereas down-regulated p-p53 in NHBE cells. The changes of typical autolysosom and representative autophagosome in the presence of CCE or/and autophagy inhibitor chloroquine (CQ) were also observed by transmission electron microscopy. These data demonstrated that CCE reduced CSE-induced autophagy flux activation in NHBE cells. The blockade of CCE on autophagy flux contributes to its protection against CSE-induced NHBE cells damage, and CCE is promising to be combination therapeutic molecules to excessive autophagic damage in respiratory diseases.

[Effective components and signaling pathways of Epimedium brevicornumbased on network pharmacology].

The aim of this paper was to find out the active components of Epimedium brevicornum using network pharmacology, and find the potential targets and mechanisms. The TCMSP database was used to screen the active ingredients, and TTD and DrugBank databases were used to predict the potential targets with the literature mining. The pathway annotation was used to enrich and analyze the active ingredients and potential targets of E. brevicornum. The results showed that E. brevicornum had34 potential target active ingredients, including 21 flavones components, such as icariin, epimedin A, epimedin B, epimedin C, Yinyanghuo A, Yinyanghuo C and so on, 2 lignans involved in (+)-cycloolivil and olivil, 3 sterols consisting of sitosterol, 24-epicampesterol and poriferast-5-en-3beta-ol. The main predicted targets included Ptgs2, NCOA6, RANK, OPG, WNT9B, PTH1R, BMPs, SMAD4A and so on. There were 88 signaling pathways involved in 10 signaling pathways which was related to inflammation, such as NF-kappa B signaling pathway, T cell receptor signaling pathway, IL-17 signaling pathway and 10 pathways which was related to cancer included breast cancer, bladder cancer, pancreatic cancer and so on, and estrogen related signaling pathways included estrogen signaling pathway. This laid the foundation for the discovery of the active components of Epimedium and the study on its mechanism of action.

SIRT1 rs10823108 and FOXO1 rs17446614 responsible for genetic susceptibility to diabetic nephropathy.

SIRT1 and FOXO1 play an important role in the pathogenesis of diabetic nephropathy (DN). However, the association between genetic polymorphisms and susceptibility to type 2 DN (T2DN) has not been explored. In this study, a total of 1066 patients with type 2 diabetes mellitus (T2DM) (413 without and 653 with DN) were enrolled. The genotypes of three htSNPs (rs3818292, rs4746720, rs10823108) within SIRT1 and two htSNPs (rs2721068, rs17446614) in FOXO1 were determined by PCR-RFLP. HbA1C, LDL, HDL, TC, and TG levels were also examined. SIRT1 rs10823108 AA genotype was significantly associated with a decreased risk of DN (OR = 0.60, 95%CI: 0.38-0.97), while GA genotype (OR = 1.77, 95%CI: 1.33-2.35) and AA genotype (OR = 2.32, 95%CI: 1.25-4.34) of FOXO1 rs17446614 was associated with an increased T2DN risk. The interactions among rs1744 6614, BMI and duration of diabetes (OR: 2.63, 95%CI: 1.23-4.31) were also observed. Subsequent haplotype analysis revealed that two haplotype defined by AC (OR: 1.50, 95%CI: 1.15-1.94) and AT (OR: 1.79, 95%CI: 1.06-2.80) within FOXO1 gene may increase the risk of T2DN. In conclusion, genetic variant rs10823108 in SIRT1 and variant rs17446614 in FoxO1 may contribute to the risk of DN in T2DM patients.

ß-elemene regulates endoplasmic reticulum stress to induce the apoptosis of NSCLC cells through PERK/IRE1α/ATF6 pathway.

Endoplasmic reticulum stress (ERs) has been regarded as an important cause for the pathogenesis of non-small-cell lung cancer (NSCLC). ß-elemene is an active component in the essential oil extracted from a medicinal herb, Curcuma wenyujin, and has been reported to be effective against non-small-cell lung cancer (NSCLC). However, the potential effect and underlying mechanisms of ß-elemene on regulating ERs to inhibit NSCLC are still unclear. In the present study, A549 cells and Lewis tumor-bearing C57BL/6J mice were established to evaluate this effect. Visualsonics Vevo 2100 Small Animal Dedicated High-frequency Color Ultrasound was performed to observe tumor volume in vivo. 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) was used to evaluate cell vitality of A549 cells. Furthermore, western blotting (WB), immunohistochemistry (IHC) and quantitative reverse transcription polymerase chain reaction (q-PCR) were applied to detect the ERs-related proteins. Flow cytometry was also applied to detect cell apoptosis and assay kit for reactive oxygen species (ROS) generation. Our results showed that ß-elemene inhibited lung cancer tumor growth and cell vitality in a dose- and time-dependent manner. Not only that, ß-elemene could up-regulate ERs-related proteins like PERK, IRE1α, ATF6, ATF4, CHOP and down-regulate the Bcl-2 expression. More importantly, ERs inhibitor 4-PBA, IRE1α inhibitor STF-083010, ATF6 inhibitor Anti-ATF6 and PERK inhibitor GSK2656157 can all reduce the amplitude of protein expression changes and apoptosis rates, then weaken the anti-tumor effect of ß-elemene. Therefore, the present in vivo and in vitro study revealed that the anti-NSCLC effect of ß-elemene is closely related to the activation of ERs through PERK/IRE1α/ATF6 pathway, and this might be beneficial for clinical therapy of NSCLC.

Alisol A 24-Acetate and Alisol B 23-Acetate Induced Autophagy Mediates Apoptosis and Nephrotoxicity in Human Renal Proximal Tubular Cells.

Two natural compounds alisol A 24-acetate (24A) and alisol B 23-acetate (23B) are abundant in Rhizoma alismatis. In the present study, we evaluated the induction of 24A and 23B on apoptosis and possible nephrotoxicity of human renal proximal tubular (HK-2) cells by activating autophagy and also explored its regulation on PI3K/Akt/mTOR signaling pathway. Presently, Clusterin, Kim-1, and TFF-3 were considered to be new bioindicators of nephrotoxicity. Interestingly, the protein expression and mRNA levels of Clusterin, Kim-1 and TFF-3 could be significantly increased by 23B and 24A in vivo and in vitro. Furthermore, cell apoptosis could be triggered by 23B and 24A via significantly decreasing the protein expression and mRNA levels of Bcl-2 and Bcl-xl. Autophagy of HK-2 cells could be induced by both 23B and 24A via significantly enhancing the ratio of LC3II/LC3I, the protein expression of Beclin-1 as well as the mRNA levels of LC3 and Beclin-1. Meanwhile, PI3K/Akt/mTOR signaling pathway could be inhibited by these two compounds. An autophagy inhibitor, 3-methyladenine, could partially reverse cell viability and conversely change the ratio of LC3II/LC3I and the protein expression of Bcl-2 and Kim-1. Thus this study helped to understand that 23B and 24A induced autophagy resulted in apoptosis and nephrotoxicity through inhibiting PI3K/Akt/mTOR signaling pathway, facilitating further studies for nephrotoxicity induced by these two compounds and could be beneficial for safe use of Rhizoma alismatis in clinic.

Successful treatment of cardiac electrical storm in dilated cardiomyopathy using esmolol: A case report.

The present study reports a case of electrical storm occurring in a 43-year-old woman with dilated cardiomyopathy. The patient suffered from a cardiac electrical storm, with 98 episodes of ventricular tachycardia rapidly degenerating to ventricular fibrillation in hospital. The patient was converted with a total of 120 defibrillations. Recurrent ventricular tachycardia/fibrillation was initiated by premature ventricular beats. The patient did not respond to the use of amiodaronum. However, the administration of esmolol stabilized the patient's heart rhythm. A moderate dose of the ß-blocker esmolol, administered as an 0.5-mg intravenous bolus injection followed by an infusion at a rate of 0.15 mg/kg/min, inhibited the recurrence of ventricular fibrillation and normalized the electrocardiographic pattern. The results suggest that esmolol may be able to improve the survival rate of patients with electrical storm in dilated cardiomyopathy and should be considered as a primary therapy in the management of cardiac electrical storms.

Rosuvastatin Attenuates CD40L-Induced Downregulation of Extracellular Matrix Production in Human Aortic Smooth Muscle Cells via TRAF6-JNK-NF-κB Pathway.

CD40L and statins exhibit pro-inflammatory and anti-inflammatory effects, respectively. They are both pleiotropic and can regulate extracellular matrix (ECM) degeneration in an atherosclerotic plaque. Statins can decrease both the CD40 expression and the resulting inflammation. However, the effects of CD40L and stains on atherosclerotic plaque ECM production and the underlying mechanisms are not well established. Moreover, prolyl-4-hydroxylase α1 (P4Hα1) is involved in collagen synthesis but its correlations with CD40L and statins are unknown. In the present study, CD40L suppressed P4Hα1 expression in human aortic smooth muscle cells (HASMCs) in a dose- and time-dependent manner, with insignificant changes in MMP2 expression and negative enzymatic activity of MMP9. CD40L increased TRAF6 expression, JNK phosphorylation, NF-κB nuclear translocation as well as DNA binding. Furthermore, silencing TRAF6, JNK or NF-κB genes abolished CD40L-induced suppression of P4Hα1. Lower NF-κB nuclear import rates were observed when JNK or TRAF6 silenced HASMCs were stimulated with CD40L compared to HASMCs with active JNK or TRAF6. Together, these results indicate that CD40L suppresses P4Hα1 expression in HASMCs by activating the TRAF6-JNK- NF-κB pathway. We also found that rosuvastatin inhibits CD40L-induced activation of the TRAF6-JNK- NF-κB pathway, thereby significantly rescuing the CD40L stimulated P4Hα1 inhibition. The results from this study will help find potential targets for stabilizing vulnerable atherosclerotic plaques.

MiR-34c-3p suppresses the proliferation and invasion of non-small cell lung cancer (NSCLC) by inhibiting PAC1/MAPK pathway.

MicroRNAs have become recognized as key players in the development of malignancy. They are a family of small non-coding RNAs (22 nt~30 nt) that can negatively regulate the expression of cancer-related genes by sequence selective targeting of mRNAs, leading to either mRNA translational repression or degradation. Lung cancer is the leading cause of cancer-related death worldwide with a substantially low survival rate. In this study, we analyzed the expression profile of miR-34c-3p in non-small cell lung cancer (NSCLC) tissues and cell lines, as its participation in some other types of cancer has been shown by previous reports. We found that miR-34c-3p was downregulated both in NSCLC tissues and cell lines. Overexpression of miR-34c-3p suppressed cell proliferation and colony formation and also limited migration and invasion in A549 cells. Furthermore, our results also shown miR-34c-3p reduction was associated with increased PAC1 expression levels in which miR-34c-3p downregulated PAC1 expression by recognizing and binding to specific binding sites in PAC1 3'-UTR. Taken together, our study implicates important roles of miR-34c-3p in NSCLC pathogenesis and implicates its potential application in cancer therapy.